The majority of excitatory neuronal input occurs at dendritic spines; remodeling of these structures leads to concurrent changes in neuronal function essential for normal neural development and processing. Furthermore, dysregulation of this phenomenon underlies numerous neurological and neurodegenerative disorders. In addition to signaling at synapses, signaling pathways to the nucleus are thought to be important for changes in dendritic spine morphology and neuronal plasticity. However, the signal transduction networks that regulate spine modulation are not well understood. AF-6 is a PDZ-domain-containing protein abundantly expressed at adhesion junctions and is involved in dendritic spine remodeling. Pathways upstream of AF-6, including NMDA receptor (NMDAR), estrogen (E2) and N-cadherin-dependent signaling, have been associated with modulating dendritic spine morphology via an AF-6 dependent pathway. In our preliminary data, we demonsrate that these pathways can also induce AF-6 translocation to the nucleus. Thus AF-6 is an ideal candidate to mediate dendritic spine morphogenesis by signaling in distinct subcellular compartments. The aim of this proposal is to determine the role of differential AF-6 translocation in regulating dendritic spine morphogenesis in cultured cortical neurons. First, we will characterize the time-dependent localization of AF-6 in NMDAR, E2 and N-cadherin-mediated signaling by using immunocytochemistry and Western-blotting techniques. We will also assess the role of the various AF-6 domains in its translocation by overexpressing truncated or mutated AF-6 constructs. Next, we will use immunocytochemistry to determine changes in spine expression induced by NMDAR, E2 and N-cadherin-mediated signaling or the overexpression of altered AF-6 constructs. Lastly, we will intervene with AF-6 translocation and then assess alterations in spine morphology. To this end, we will prevent AF-6 from localizing to synapses or the nucleus using specifically modified AF-6 constructs and we will force AF-6 into the nucleus using a nuclear localization signal. Results from these studies will foster a better understanding of the signaling pathways regulating dendritic spine morphogenesis and provide insight into the mechanisms governing synaptic plasticity in health and disease.